| 'Zang-Yin-Chen'(ZYC) is a traditional Tibetan medicine for the activity of hepatoprotective effect, antiinflammatory, antioxidative damage. It is suggested by phytochemistry study that iridoids,xanthones and triterpenoids are the mainly components in ZYC. In recent years, the compounds of xanthones and iridoids were studied widely. But the activity of these compouds were not good. For the purpose of new active substance discovery, the activity compounds of ZYC was modified by biology methods.The main research works are as follows:With the average transfer rate of swertiamarin and swertianolin as index, the best extraction technology of these constituents in ZYC was optimized by orthogonal experimental design. And macroporous resin, silica gel column chromatography were used for the separation of swertiamarin,swertianolin. And the purity is more than 80%.The utilization of swertiamarin,swertianolin by Aspergillus niger was investigated. And result showed these compouds could be transformed by it. For the purpose of compounds preparation, swertiamarin was coincubated with Aspergillus niger. The compouds including sweroside and (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c] pyridin-1-one were prepared. The medium was optimizated for the output of EHPO. The optimizated medium composition was glucose 5 g/L; Tryptone 7.5g/L; KH2PO45 g·/L; MnSO4 1.51 g/L; MgSO4 1.21 g/L, NaCl 0.59 g/L, pH of the medium was adjusted to 6.0.With the transfer rate of EHPO as index, the separation technology of EHPO was established. The fermentation broth was concentrated under reduced pressure and partitioned with petroleum (60-90℃). The aqueous layer was isolated by macroporous resin column chromatography. And the column was eluted with H2O (3BV),10% ethanol (3BV) followed by 50% ethanol (3BV). The faction eluted by 50% ethanol was isolated by Al2O3 column chromatography. And the column was eluted with acetoacetate-methanol (8:2) solution (9BV), acetoacetate-methanol (6:4) solution (4BV), followed by acetoacetate-methanol (2:8) solution (12BV). Finaly, the compounds such as EHPO and swertiamarin were isolated by preparative HPLC. The purity of them is more than 95%.In the Caco-2 cell model, the PappAP-BL of the EHPO was equal to the PappBL-AP, and Papp keep almost constant within the concentration investigated. The permeability of EHPO is good. In Caco-2 cell model, the transcellular transport mechanism of it mainly is a passive one. But the Papp still could be effected by pH, Verapamil.The antioxidative property of EHPO was evaluated by malondialdehyde (MDA) formation in a hepatic homogenates model, and a model of H2O2-induced hemolysis. The result showed EHPO could inhibit hepatic homogenate peroxidation. In the model of H2O2-induced hemolysis model, it could inhibit hemolysis significantly(0.8mg/mL). (P<0.05).The hepatoprotective effect was evaluated by the model of CCl4 induced acute hepatic injure in mice. EHPO could decrease the serum ALT, AST levels, the concentration of MDA and increase SOD activity in the mice treated with CCl4. It exhibited significant hepatoprotective effect.In summary, the results showed that swertiamarin and swertianolin could be transformed by Aspergillus niger. The technology we set could separate the precurosor compounds and the product. The compound of EHPO yielded by co-incubated swertiamarin with Aspergillus niger could be absorbed soon. And it has the activity of antioxidative, hepatoprotective effect.
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